Alkaline phosphatase tryptophan

The phosphorescence lifetimes of various proteins at room temperature are given in Table 3.1. Some variability in the lifetimes reported from lab to lab is evident, possibly due to different enzyme preparation, removal of oxygen (see below), or other conditions. Nevertheless, when measured under the same conditions, it is apparent that the tryptophan lifetimes vary dramatically from protein to protein. Alkaline phosphatase exhibits the longest lifetime from a protein in solution with a lifetime of 1.5—1.7 s at 22°C, approaching the lifetime of 5.5 s at 77 K. The lifetime of free indole in solution is 15—30 /is at 22°C.(38 39) Therefore, in the absence of other quenching mechanisms, the lower limit for the phosphorescence lifetime of a fully exposed tryptophan moiety in a protein should be about 20 /is. 

Kai and Imakubo(76) found that the temperature at which emission from the exposed tryptophan is no longer observed appears to be characteristic of the protein, having values of 180 K for trypsin, 200 K for aldolase, and 230 K for alkaline phosphatase. 

The long-lived phosphorescence of the tryptophan in alkaline phosphatase is unusual. Horie and Vanderkooi examined whether its phosphorescence could be detected in E. coli strains which are rich in alkaline phosphatase.(89) They observed phosphorescence at 20°C with a lifetime of 1.3 s, which is comparable to the lifetime of purified alkaline phosphatase (1.4 s). Long-lived luminescence was not observed from strains deficient in alkaline phosphatase. The temperature dependence of tryptophan phosphorescence in the living cells was slightly different from that for the purified enzyme, indicating an environmental effect. 

Isolation of alkaline phosphatase from Escherichia coli in which 85% of the proline residues were replaced by 3,4-dehydro-proline affected the heat lability and ultraviolet spectrum of the protein but the important criteria of catalytic function such as the and were unaltered (12). Massive replacement of methionine by selenomethionine in the 0-galactosidase of E. coli also failed to influence the catalytic activity. Canavanine facilely replaced arginine in the alkaline phosphatase of this bacterium at least 13 and perhaps 20 to 22 arginyl residues were substituted. This replacement by canavanine caused subunit accumulation since the altered subunits did not dimerize to yield the active enzyme (21). Nevertheless, these workers stated "There was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine." An earlier study in which either 7-azatryptophan or tryptazan replaced tryptophan resulted in active protein comparable to the native enzyme (14). 

Fiqtire 3.5 (a) Competitive inhibition inhibitor and substrate compete for the same binding site. For example, indole, phenol, and benzene bind in the binding pocket of chymotrypsin and inhibit the hydrolysis of derivatives of tryptophan, tyrosine, and / phenylalanine, (b) Noncompetitive inhibition inhibitor and substrate bind simultaneously to the enzyme. An example is the inhibition of fructose 1,6-diphosphatase by AMP. This type of inhibition is very common with multisubstrate enzymes. A rare example of / uncompetitive inhibition of a single-substrate enzyme is the inhibition of alkaline phosphatase by L-phenylalanine. This enzyme is composed of two identical subunitjs, so presumably the phenylalanine binds at one site and the substrate at the other. [From N. K. Ghosh and W. H. Fishman, J. Biol. Chem. 241, 2516 (1966) see also M. Caswell and M. Caplow, Biochemistry 19, 2907 (1980). 

Photooxidation of alkaline phosphatase in the presence of methylene blue and Rose Bengal causes loss of activity for both native and apo-enzyme. In the case of the native enzyme, zinc protects 2 to 3 of the 16 histidine residues. The rate of oxidation of tryptophan is not affected by zinc, and there was no loss of tyrosine. Also, photooxidation of the apoenzyme diminishes zinc binding. It would appear that histidine residues play a role in binding the two zinc ions necessary for enzymic activity (91). 

Treatment of the enzyme with A-bromosuccinimide oxidized 2 of the 8 tryptophan residues and 8 of the 20 tyrosine residues, but none of the histidine residues. This treatment causes the phosphotransferase activity with tris as an acceptor to double and the hydrolase activity to increase slightly. In the case of cobalt alkaline phosphatase, the above treatment caused a threefold increase in hydrolase activity and the generation of an even greater phosphotransferase activity (91). 

Krstulovic and co-workers (K30) reported on the development of assays for serum acid and alkaline phosphatase enzymes. These enzymes have been reported to be elevated in various disease states (A2, B14, F2, Kl). In addition, they developed a method for tryptophanase analysis using RPLC (K29). Employing fluorometric detection, the substrate, tryptophan, and reaction product, indole, can be monitored selectively with high sensitivity. They reported on several advantages of this method, including minimal sample preparation, rapid analysis time, and high specificity. 

The human venous plasma and whole blood contain amino acids and the successful identification and quantification of those amino have been previously reported [28, 29]. Conconi et al. [30] reported that the amino adds (lysine, threonine, methionine, tryptophan, arginine, which are all present in the DMEM solutions) increased both the osteoblast proliferation and alkaline phosphatase activity of rat osteoblasts cultured in vitro. Imamura et al. [31] and Tentorio and Canova [32] separately showed that the amino acid lysine adsorbs itself on pure metallic Ti and on amorphous Ti hydrous oxide surfaces, respectively, at neutral pH values. While the inoiganic SBF solutions cannot provide any practical means of producing synthetic biomaterials with some amino acids adsorbed on their surfaces, DMEM solutions can provide unique biomaterial surfaces already containing adsorbed amino acids. 

The postnatal development of alkaline phosphatase in the intestinal mucosa of rats and mice is similar to that of tryptophan pyrrolase. Much work in mammalian developmental biochemistry was done on developing liver. Studies on prenatal liver include investigations on the bioenergetic pathways and studies of the formation of enzyme found exclusively in liver. Burch [13] measured the activity of several enzymes involved in glycolysis, the hexose monophosphate shunt, glycogenolysis, and gluconeogenesis. The results of these studies show that the biochemical development of the liver can be divided into three periods prenatal, from 0 to 21 days, and after 21 days. 

Some physiological or pathological stimuli induce the liver cell to synthesize or break down some protein selectively. Even during starvation the content of all liver protein does not drop simultaneously. For example, while the activities of catalase, xanthine oxidase, alkaline phosphatase, and acid phosphatase drop at various rates as starvation progresses, that of glucose-6-phosphatase increases. Hydrocortisone and tryptophan administration induces a massive increase in tryptophan peroxidase activity. In either case, at least part of the increase in enzyme activity results from de novo enzyme synthesis. If tryptophan administration is interrupted, the activity of the peroxidase returns to normal. During the induction, turnover rates of other proteins do not change. 

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